Wild mouse species and the various inbred laboratory mouse strains differ from one another in their susceptibility to the mouse gammaretroviruses and retrovirus-induced cancers. These differences are due to variations in specific host genes, and we have been engaged in an ongoing effort to identify and characterize several mouse genes involved in virus resistance. Our major interest has been on factors that interfere directly with virus infection and replication, and we focus our efforts on those factors that inhibit virus entry and the early post-entry stages of the virus replicative cycle. At the level of entry, there are two types of resistance genes that target the receptor-virus interaction. Receptors can be blocked by virus envelope glycoprotein produced by endogenous retroviruses, or resistance can be caused by polymorphisms in the cell surface receptor. After the gammaretrovirus enters the receptive cell, reverse transcription and translocation to the nucleus can be inhibited or altered by virus resistance factors Fv1, mApobec/Rfv3, and TRIM5alpha. Our current aim is to characterize these resistance factors and the viruses they target, define the origin and extent of antiviral activity in Mus evolution, and elucidate the responsible mechanisms. This work relies heavily on wild mice because laboratory strains provide only a limited sampling of the genetic diversity in Mus. Also, wild mouse species allow us to examine survival strategies in natural populations that harbor virus and to follow the evolution of the resistance genes. These mice additionally provide a source of novel resistance genes and virus variants. One set of projects aims to identify viral and cell receptor determinants responsible for virus binding and entry. We are currently working on the XPR1 receptor for the xenotropic/polytropic mouse gammaretroviruses (XP-MLVs) and for XMRV, a xenotropic virus-like virus isolated from humans with prostate cancer or chronic fatigue syndrome. We have determined that, in mouse populations exposed to infectious virus, virus resistance is mediated by polymorphisms of the cell surface receptor. We have now identified a total of four XPR1 susceptibility variants in wild mice and described the geographic and species distribution of the Mus Xpr1 variants. Three of the four receptors restrict entry by two or more of the virus host range variants that rely on XPR1, and all three of these receptors evolved in populations exposed to X-MLVs. We used mutagenesis and phylogenetic analysis to evaluate the functional contributions made by conserved, variable and deleted residues in this receptor. Rodent Xpr1 is under positive selection indicating a history of host-pathogen conflicts;several codons under selection have known roles in virus entry. All non-Mus mammals are susceptible to mouse X-MLV, but some restrict other members of this family of viruses and the resistance of hamster and gerbil cells to the human-derived XMRV indicates that XMRV has unique receptor requirements. We also showed that the hypervariable fourth extracellular XPR1 loop (ECL4) contains 3 evolutionarily constrained residues that do not contribute to receptor function. We also identified two novel residues important for virus entry, and we described a unique pattern of variation in the 3 virus-restrictive Xpr1 variants found in MLV-infected house mice;these mice carry different deletions in the receptor determining region of XPR1 suggesting either that these sites or loop size affect receptor function. We also determined that the XPR1 receptor variant found in the laboratory mouse is not found in wild mouse populations. This laboratory mouse allele encodes a receptor that is uniquely resistant to xenotropic gammaretroviruses. We screened laboratory mouse strains for sequence and functional variants of the XPR1 receptor, and identified the permissive wild mouse variant of this receptor in 7 laboratory strains. We also typed these strains for Bxv1, an endogenous germline copy of the xenotropic virus that is capable of producing infectious virus. We identified Bxv1 in many strains of mice, and traced its ancestry to the Japanese wild mouse, M. m. molossinus. Bxv1 and the permissive allele were found together in one strain, F/St, which is characterized by lifelong viremia with xenotropic virus. In another series of experiments, we have been using phylogenetic methods to identify and characterize host genes that have had an anti-viral role in the genus Mus. Among the mouse genes responsible for resistance to mouse leukemia viruses is Rfv3 (recovery from Friend virus). This gene was recently shown to be encoded by the mouse APOBEC (mA3) gene, a cytidine deaminase gene known to restrict other retroviruses in mice and in humans. We sequenced mA3 from multiple laboratory and wild mice to examine its evolution. We discovered that the mA3 allele in virus resistant mice is disrupted by insertion of the regulatory signals of a mouse leukmia virus that correlated with enhanced mA3 expression in virus resistant mouse strains and species. We also identified sites in mA3 under positive selection that specify residues in two loops along the groove that forms the mA3 active site. We demonstrated by mutagenesis and functional assays that one of those two loops affects mA3 antiviral activity. We thus showed that mA3 has had an antiviral role throughout mouse evolution, and we identified an inserted regulatory sequence and two clusters in the protein coding sequence that contribute to this antiviral function. More recently we focused on the fact that mA3 transcripts have different splicing patterns in virus-resistant mice like C57BL/6 compared with virus-sensitive strains like BALB/c. C57BL/6 transcripts lack exon 5;this exon is spliced into mA3 of BALB/c and separates the two cytidine deaminase domains. We have now showed tht mA3 exon 5 is a functional element that influences protein synthesis at a post-transcriptional level. We also used in vitro splicing assays to identify two critical polymorphisms affecting the inclusion of exon 5 into mA3 transcripts: the number of TCCT repeats upstream of exon 5 and a single nucleotide substitition within exon 5. We also found that distribution of exon 5 into mA3 mRNA is a relatively recent event in the evolution of mice. The widespread geographic distribution of the exon 5-including genetic variant suggests that in some Mus populations the cost of maintaining an effective but mutagenic enzyme may outweigh its antiviral function.